RESUMEN
The ability of 2-deoxy-d-glucose (2-DG) to interfere with d-glucose metabolism demonstrates that nutrient and energy deprivation is an efficient tool to suppress cancer cell growth and survival. Acting as a d-glucose mimic, 2-DG inhibits glycolysis due to formation and intracellular accumulation of 2-deoxy-d-glucose-6-phosphate (2-DG6P), inhibiting the function of hexokinase and glucose-6-phosphate isomerase, and inducing cell death. In addition to glycolysis inhibition, other molecular processes are also affected by 2-DG. Attempts to improve 2-DG's drug-like properties, its role as a potential adjuvant for other chemotherapeutics, and novel 2-DG analogs as promising new anticancer agents are discussed in this review.
Asunto(s)
Desoxiglucosa/análogos & derivados , Desoxiglucosa/uso terapéutico , Glioblastoma/tratamiento farmacológico , Muerte Celular/efectos de los fármacos , Terapia Combinada , Desoxiglucosa/química , Desoxiglucosa/farmacología , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Glucosa/química , Glucosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/metabolismo , Glucólisis/efectos de los fármacos , Hexoquinasa/metabolismo , HumanosRESUMEN
Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea extract (GTE) that was widely believed to reduce proliferation of many cancer cell lines. The purpose of this study was to verify the possible pro-apoptotic action of GTE/EGCG in human colon adenocarcinoma COLO 205 cells. The effect of EGCG/GTE treatments on cell viability was studied using methyl thiazolyl tetrazolium (MTT) assay. Cell proliferation was assessed with crystal violet staining, whereas protein expression levels were evaluated by western blotting followed by densitometric analysis. Obtained results were analyzed statistically. Surprisingly, EGCG/GTE dose-dependently up-regulated COLO 205 cells viability and proliferation. Observed effects were mediated by lipid rafts, as cholesterol depletion significantly prevented EGCG/GTE-dependent cell survival. Furthermore, treatment of COLO 205 cells with EGCG/GTE resulted in activation of MEK/ERK1/2, but not Akt1/2/GSK-3ß signaling pathway. The presence of MEK inhibitor - PD98059 but not PI3-K inhibitor - LY294002, both reduced EGCG/GTE-induced ERK1/2 activation and the proliferative effect of catechins. Furthermore, EGCG/GTE stimulated secretory clusterin (sClu) expression level, which underwent complex control through lipid rafts/PKC/Wnt/ß-catenin system. Our studies demonstrated that EGCG and GTE stimulate cell survival and proliferation of COLO 205 cells in a lipid rafts-dependent manner via at least MEK/ERK1/2 signaling pathway. Furthermore, EGCG/GTE mediated positive effects on viability and mitogenicity of COLO 205, while suppression of ß-catenin activity was positively correlated with sClu clusterin expression.
Asunto(s)
Camellia sinensis/química , Catequina/análogos & derivados , Microdominios de Membrana/efectos de los fármacos , Extractos Vegetales/farmacología , Catequina/farmacología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Clusterina/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microdominios de Membrana/enzimología , Microdominios de Membrana/metabolismo , Factores de Tiempo , beta Catenina/antagonistas & inhibidoresRESUMEN
Transient treatment of human adenocarcinoma COLO 205 cells with lipit raft (LR) modulators (MßCD, NY, IMP) was followed by the challenge with metabolic inhibitors and selected anti-cancer drugs. To overturn cholesterol chelation, the MßCD, NY treatment was followed by cholesterol conjugates (CHOL-MßCD or CHOL-PEG). The TNF-α- and P(Ser473)-PKB/Akt1/2-mediated effects initiated at LR were evaluated with regard to cell viability and mitogenicity. Cholesterol chelators reversibly reduced cell survival, whereas some of the tested compounds had weak effects (CIS, CLA), stimulated (EGCG) or reduced (NaB) cell survival. Cellular localizations of LR-associated molecules (ceramides, Gαi-2 heterotrimeric protein, and TNF-R1) in different cellular compartments including the plasma membrane were observed in the respective photographs from TEM and SEM. Evidence from SEM also showed that TNF-R1 is clustered on the surface of COLO 205 cells without presence of cognate ligand but clustering is promoted by TNF-α, while it vanished after IMP treatment. COLO 205 cells remained immune to TNF-α-induced apoptosis unless NaB was added, in which case NaB-induced cell death was further potentiated by TNF-α. Combined NaB and TNF-α treatment was associated with marked changes in the expression of pro- and antiapoptotic proteins. In this study, we demonstrated that initial excess of prosurvival signals could be diminished by cholesterol chelators, whereas LR-independent cell survival could be targeted by NaB. Apparently, lipid rafts do not participate in NaB-dependent cell death.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Butiratos/farmacología , Colesterol/fisiología , Neoplasias del Colon/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adenocarcinoma/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Colesterol/farmacología , Neoplasias del Colon/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Humanos , Imipramina/farmacología , Invasividad Neoplásica , Sodio/química , Sodio/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidoresRESUMEN
Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not influence Vero cell cytoskeleton. Conversely, the Jan-E strain induced polymerization of both F-actin and MT in Vero cells, but not in ED cells. Confocal-microscopy analysis revealed that alpha-tubulin colocalized with viral antigen in ED cells infected with either Rac-H or Jan-E viruses. Alterations in F-actin and alpha-tubulin were evaluated by confocal microscopy, Microimage analysis and scanning cytometry. This unique combination allowed precise interpretation of confocal-based images showing the cellular events induced by EHV-1. We conclude that examination of viral-induced pathogenic effects in species specific cell lines is more symptomatic than in heterologous cell lines.
Asunto(s)
Citoesqueleto/química , Herpesvirus Équido 1/patogenicidad , Actinas/metabolismo , Animales , Apoptosis , Chlorocebus aethiops , Citometría de Barrido por Láser , Microscopía Confocal , Piel/citología , Piel/virología , Células VeroRESUMEN
Bisindolylmaleimide derivatives were originally described as protein kinase C inhibitors. However, several studies have shown that bisindolylmaleimides target several other signaling molecules. The review presents bisindolylmaleimide-mediated PKC-dependent and PKC-independent biological effects, such as reversal of MDR and modulation of Wnt signaling through GSK-3b and b-catenin. Importantly, the potent proapoptotic properties of bisindolylmaleimides are also described. Bis-IX appears as the most efficient activator of intrinsic apoptotic pathway and additionally, facilitates extrinsic apoptosis. Presented molecular mechanisms indicate that bisindolylmaleimides could be useful agents in anticancer therapy. They repress uncontrolled proliferation and restore the sensitivity to chemotherapy which allows eradication of cancer cells.
Asunto(s)
Indoles/uso terapéutico , Maleimidas/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Humanos , Neoplasias/enzimologíaRESUMEN
There is no universal approach to stop muscle cachexia in a number of life-threatening diseases. Accordingly, it is uncertain why the body mass is so critical to keep alive patients with cancer, congestive heart failure (CHF), AIDS or sepsis. At present, it is widely believed that excess muscle wasting diminishes lean body mass to the risky level accompanied by anorexia, anemia, lipolysis, acute phase response and insulin resistance. If missed and/or untreated muscle cachexia inevitably leads to death due to cardiac and respiratory failure (almost one-third of all cancer deaths). This complex metabolic disorder is suited by the elevated levels of inflammatory cytokines (TNF-alpha, IFN-gamma, IL-1-beta, IL-6, IL-2) and low levels of anti-inflammatory/ other cytokines (IL-15, leptin). Concurrently, tissue sensitivity to insulin is considerably reduced. Recent findings indicate that entirely few muscle-specific genes (i.e. MyoD and myosin heavy chain, MyHC) and their products must be targeted to initiate muscle wasting. Muscle atrophy occurs at different levels, starting from repressed gene expression and ended with accelerated protein degradation. Muscle growth (myogenesis) is severely compromised and disruption of sarcomere architecture heralds the proteolysis of contractile apparatus. This review aims to synthesize our present knowledge of intracellular mechanisms and molecular regulation of muscle cachexia with respect to cytokine signaling.
Asunto(s)
Caquexia/fisiopatología , Citocinas/metabolismo , Transducción de Señal , Animales , Caquexia/etiología , Caquexia/terapia , Regulación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina , Desarrollo de Músculos , Atrofia Muscular/etiologíaRESUMEN
COLO 205 colon adenocarcinoma cells are highly resistant to extrinsic apoptosis induced by immunomodulatory cytokines. One of the antiapoptotic mechanisms is the expression of cFLIP protein, which inhibits TNF-alpha-induced cell death. The use of metabolic inhibitors, such as sodium butyrate (NaBt), the potent repressor of histone deacetylase, sensitizes tumor cells to TNF-alpha-mediated apoptosis. The Western-blot analysis revealed that in COLO 205 cells the susceptibility to apoptogenic stimuli results from time-dependent reduction in cFLIP(L) protein assembled with DISC complex. At the same time, the level of transmembrane TNF-alpha receptor 1 (TNF-R1) was elevated which is consistent with the exaggerated rate of cell death. Since preincubation of COLO 205 cells with N-acetyl-L-cysteine (NAC), or sodium ascorbate (ASC) did not protect cells from combined NaBt- and TNF-alpha-induced apoptosis, we concluded that deletion of cancer cells is not evoked by oxidative stress. Our results suggest that the combination of TNF-alpha with NaBt targets antiapoptotic protein(s) and may provide efficient and non-toxic treatment of colon cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Butiratos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/farmacología , Adenocarcinoma/patología , Western Blotting , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/patología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos , Quimioterapia Combinada , Humanos , Estrés Oxidativo , Receptores Tipo I de Factores de Necrosis Tumoral/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de TiempoRESUMEN
One of the most promising strategies in colon cancer therapy is the sensitization of cancer cells to natural proapoptotic cytokines, such as death ligands and interferons, which are able to eliminate abnormal cells. The investigation of mechanisms determining the immune escape of cancer cells revealed the presence of antiapoptotic proteins, such as cFLIP, which inhibit cell death signal transduction. Numerous studies showed that the use of different metabolic inhibitors, such as cycloheximide (CHX), reduces the cFLIP protein level, thus restoring the susceptibility to TNF-alpha-induced apoptosis. However, high non-specific toxicity of CHX excludes the clinical use of this substance. The current efforts are focused on identification of bioactive compounds which could safely support immunotherapy. The review presents in vitro and in vivo evidence that butyrate (Bt), fatty acid produced in colon during fermentation process and parthenolide (PN), sesquiterpene lactone isolated from Tanacetum parthenium specifically affect different cancer cells. Among described various molecular mechanisms of Bt and PN action, one reduces the level of antiapoptotic proteins. This paper clearly demonstrates that bioactive compounds, especially combined with immune cytokines could be seriously considered as an alternative for routine colon anti-cancer therapy.
Asunto(s)
Butiratos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Inmunoterapia , Sesquiterpenos/uso terapéutico , Neoplasias del Colon/patología , Ácidos Grasos , Humanos , Fitoterapia , Tanacetum partheniumRESUMEN
This review outlines the molecular events that accompany the antitumor action of sodium butyrate (NaBt). Butyrate, a low-molecular weight four-carbon chain volatile fatty acid (VFA) has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate, that this product of large bowel microbial fermentation of dietary fiber, might evoke the above-mentioned effects by indirect action on genes. NaBt was shown to inhibit histone deacetylase activity, allowing DNA binding of several transcription factors. Higher genomic activity leads to the higher expression of proapoptotic genes, higher level of their protein products and elevated sensitivity to death ligand-induced apoptosis. Cancer cells might be arrested in G1 phase of cell cycle in a p21-dependent manner. Proapoptotic activity of NaBt includes higher expression of membrane death receptors (DR4/5), higher level and activation of Smad3 protein in TGF-beta-dependent apoptotic pathway, lower level of antiapoptotic proteins (cFLIP, XIAP) and activation ofproapoptotic tBid protein. Thus, both intrinsic and extrinsic apoptotic pathways are stimulated to ampify the apoptotic signals. These effects are specific for tumor but not for regular cells. Unique properties of NaBt make this agent a promising metabolic inhibitor to retard tumorigenesis to suppress tumor growth.
Asunto(s)
Apoptosis , Butiratos/farmacología , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Neoplasias/patología , Sodio/farmacología , Antineoplásicos/farmacología , Ciclo Celular , ADN/química , Ácidos Grasos/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Sistema Inmunológico , Ligandos , Modelos Biológicos , Factor de Transcripción Sp1/metabolismoRESUMEN
This review describes some aspects of uncontrolled tumor growth and development. In the past, it has been shown that colon adenocarcinomas use several tactics to avoid cell deletion and to maintain cell viability. In particular, colorectal cancer cells resist death ligands-induced apoptosis by expressing anti-apoptotic proteins. By direct interaction with FADD, the FLIP protein inhibits the signal transmission from death receptors to their cytoplasmic targets in COLO 205 cells. Colorectal cancer cells also stimulate own survival by inhibiting cytotoxic signals induced by interferons. Moreover, IFN-alpha increases immune-resistance of colon cancer cells by activation of NF-kappaB. Additionally, the cytoplasmic retention of proapoptotic protein clusterin also supports viability of cancer cells. Upon suitable stimulation normal cells are featured by clusterin translocation to the nucleus with concomitant cell death. We found that proapoptotic activity of clusterin is dependent on calcium ions, and depletion of intracellular calcium caused extensive death of COLO 205 cells. Other type of strategy to inhibit chemotherapy-dependent cell death is the activity of multidrug resistance proteins (MDR). These cell membrane efflux pumps actively expel the drugs from the cell interior to prevent their action on intracellular targets. The reversal of P-gp efflux pump in chemoresistant COLO 320 cell line was observed upon phenothiazine derivatives. The variety of antiapoptotic mechanisms in colorectal cancer cells makes anticancer therapy a great challenge but detailed knowledge of their complexicity gives promise to sensitize cancer cells to death stimuli.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Antineoplásicos/uso terapéutico , Apoptosis/inmunología , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Humanos , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Modelos BiológicosRESUMEN
Current efforts are focused on revealing the cellular factors that determine the "immune escape" of cancer cells. As an example in our study human colorectal cancer cell line COLO 205 was resistant to TNF-alpha - a death inducing ligand. Nonetheless, co-incubation TNF-alpha (10 ng/ml) with cycloheximide (5 micro g/ml, CHX) caused time-dependent (6, 12, 24 hours) cell death even though, at that concentration cycloheximide did not exert cytotoxic effect unless 24 hour of treatment. After additional pretreatment it is concluded that CHX sensitizes cells to TNF-alpha-induced apoptosis. The aim of this study was to investigate the role and properties of cellular proteins that granted COLO 205 cells survival as well as to assess the effect of several metabolic inhibitors on cell viability at the above-mentioned time-points. PKCs inhibitor staurosporine (5 microM) did not influence, whereas cPKC activator PMA (100 microM) prevented TNF-alpha-induced apoptosis in the presence of CHX. Although EDTA (2 mM) and to a lesser degree EGTA (5 mM) were individually cytotoxic, they exerted protective effect at 6 and partially at 12 hour of combined TNF-alpha and CHX treatment. Ionophore A23187 (1 microM) protected cells against cell death at 12 and 24 but only partially at 6 hour of treatment. On the other hand, AD (10 ng/ml) acted synergistically with TNF-alpha and CHX at 6 and 12 hour. It appears that resistance of COLO 205 cells is determined on genomic level by a few reaction steps in which both Ca(2+)-dependent and antiapoptotic proteins are involved. Further studies revealed that CHX treatment reduces the level of FLIP but not other members of TNF-alpha-dependent death signalosome.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Neoplasias Colorrectales/patología , Cicloheximida/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Calcimicina/farmacología , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Quelantes/farmacología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Dactinomicina/farmacología , Sinergismo Farmacológico , Ácido Edético/farmacología , Ácido Egtácico/farmacología , Activación Enzimática , Activadores de Enzimas/farmacología , Humanos , Ionóforos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Escape del TumorRESUMEN
"Immune escape" is a crucial instrument used by carcinoma cells to overcome numerous strategies of immune system to delete transformed cells. Cellular factors that make cancer cells immune to defence mechanisms are incompletely understood while some remain ambiguous. Up to date evidence points to some proteins and/or signaling molecules that might be a basis for unusual behavior of cancer cells. In particular STAT kinases are currently in the main focus of attention since they were both shown to accelerate and/or to inhibit apoptosis. In our studies we observed that human colorectal COLO 205 cancer cells were resistant to TNF-alpha- or cycloheximide-induced cytotoxicity. However, when TNF-alpha (10 ng/ml) has been given along with cycloheximide (5 micro g/ml, CHX) COLO 205 cells died extensively from apoptosis. Apparently, cycloheximide sensitized cells to TNF-alpha-induced programmed cell death. To investigate the role of STAT-1 alpha in CHX-mediated TNF-alpha-induced COLO 205 cell death certain polyphenolic compounds were studied if they modulate STAT-1 alpha phosphorylation status and STAT-1 alpha-protein interaction at the level of TNF-alpha signalosome in the 6(th), 12(th), and 24(th) hour of experiment. Neither of phenolic compound, namely PI-3K inhibitor (LY294002, 20 microM) nor MEK inhibitor (PD98059, 50 microM), nor flavonol quercetin or kaempferol (10, 100 microM) in contrast to apigenin (20 microM) influenced COLO 205 cell viability during individual or combined treatment with TNF-alpha and CHX. We conclude, that some antiapoptotic proteins were involved but not STAT-1 alpha kinase to resist TNF-alpha-dependent cell death promoting activity. Summing up, except apigenin, the above-mentioned polyphenolic compounds were unable to modulate survival signal in COLO 205 cells initially believed to be suppressed by STAT-1 alpha.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/patología , Cicloheximida/farmacología , Flavonoides/farmacología , Factor 3 de Genes Estimulados por el Interferón/metabolismo , Fenoles/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Apigenina/farmacología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Quempferoles/farmacología , Ligandos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Polifenoles , Inhibidores de Proteínas Quinasas/farmacología , Quercetina/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de Tiempo , Escape del TumorRESUMEN
Ischemia/reperfusion during liver transplantation triggers a complex cascade of inflammatory events that may lead to organ dysfunction. Herein, we investigated the consequences of hepatic ischemia/reperfusion on liver dendritic cells. Liver damage was documented by increased levels of serum alanine aminotransferase and by histopathology showing large areas of hepatocyte cytolysis. MHC class II+ CD45-B220 F4/80 dendritic cells were detected in necrotic areas 20 hours after reperfusion. Dendritic cells freshly isolated from reperfused livers displayed a mature phenotype characterized by upregulated expression of B7 costimulatory molecules; MHC-class II, and CD1d molecules. As shown by real-time PCR, IL-10, and TGF-beta mRNA accumulated in liver dendritic cells isolated after reperfusion, whereas IL-12p40 mRNA levels were decreased and IFN-gamma mRNA levels were unchanged. These results suggest that hepatic ischemia/reperfusion results in maturation of dendritic cells, which preferentially produce inhibitory cytokines.
Asunto(s)
Células Dendríticas/inmunología , Hígado , Daño por Reperfusión/inmunología , Alanina Transaminasa/análisis , Animales , Femenino , Antígenos de Histocompatibilidad Clase II/análisis , Hígado/irrigación sanguínea , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Necrosis , Daño por Reperfusión/patologíaRESUMEN
Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8alpha expression. We have studied the phenotype and maturation state of mouse splenic CD8alpha(-) and CD8alpha(+) DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8alpha(-) and CD8alpha(+) DCs into potent and equally efficient activators of naïve CD4(+) and CD8(+) T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.
Asunto(s)
Antígenos CD8/análisis , Células Dendríticas/citología , Traslado Adoptivo , Animales , Presentación de Antígeno , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Dendríticas/clasificación , Células Dendríticas/efectos de los fármacos , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunofenotipificación , Lipopolisacáridos/farmacología , Activación de Linfocitos , Antígeno de Macrófago-1/análisis , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/inmunología , Proteínas Recombinantes/farmacología , Bazo/citología , Bazo/inmunologíaRESUMEN
AIMS: To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. METHODS: This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. RESULTS: Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessles. CONCLUSIONS: These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.
Asunto(s)
Células Dendríticas/fisiología , Escherichia coli/inmunología , Inmunohistoquímica/métodos , Fijación del Tejido/métodos , Animales , Células Dendríticas/citología , Femenino , Inmunidad Celular/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Coloración y Etiquetado , Linfocitos T/fisiologíaRESUMEN
The induction of immune responses in vivo is typically performed with antigens administered in external adjuvants, like alum, complete Freund's adjuvant, LPS and, more recently, monophosphoryl lipid A (MPL). However, the role of the adjuvant is still poorly defined. The aim of this study was to test whether the MPL affects the function of antigen-presenting cells (APC) in vitro and in vivo. Antigen-pulsed APC [including macrophages, B cells and dendritic cells (DC)] were incubated or not with MPL, and their ability to sensitize naive T cells was tested in vitro and in vivo. The data show that MPL enhances the ability of macrophages and B cells to sensitize naive T cells, and confers to them the capacity to induce the development of T(h)1 and T(h)2. Administration of MPL i.v. in mice results in the redistribution of fully mature DC in the T cell area of the spleen. These observations suggest that MPL may induce an antigen-specific primary immune response by provoking the migration and maturation of DC that are the physiological adjuvant of the immune system.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Presentadoras de Antígenos/efectos de los fármacos , Lípido A/análogos & derivados , Animales , Linfocitos B/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/fisiología , Femenino , Lípido A/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB CRESUMEN
Antibodies against CD3epsilon are widely used as immunosuppressive agents. Although it is generally assumed that these reagents exert their immunomodulatory properties by inducing T-cell deletion and/or inactivation, their precise mechanism of action remains to be elucidated. Using a murine model, we demonstrate in this report that administration of anti-CD3epsilon antibodies causes the migration and maturation of dendritic cells (DC) in vivo, as determined by immunohistochemical analysis. This maturation/migration process was followed by selective loss of splenic DC, which resulted in a selective inhibition of antigen-presenting cell (APC) functions in vitro. Spleen cells from anti-CD3epsilon-treated animals were unable to productively stimulate naive alloreactive T cells and Th1-like clones in response to antigen, while retaining the ability to present antigen to a T-cell hybridoma and Th2 clones. Anti-CD3epsilon treatment was found to induce a selective deficiency in the ability of spleen cells to produce bioactive interleukin-12 in response to CD40 stimulation. APC dysfunction was not observed when nonmitogenic forms of anti-CD3epsilon antibodies were used, suggesting that splenic DC loss was a consequence of in vivo T-cell activation. Nonmitogenic anti-CD3epsilon monoclonal antibodies were found to be less immunosuppressive in vivo, raising the possibility that APC dysfunction contributes to anti-CD3epsilon-induced immunomodulation. Collectively, these data suggest a novel mechanism by which mitogenic anti-CD3epsilon antibodies downregulate immune responses.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Presentación de Antígeno/inmunología , Complejo CD3/inmunología , Terapia de Inmunosupresión , Linfocitos T/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Ratones , Inmunología del TrasplanteRESUMEN
Data from adoptive transfer of mature dendritic cells (DC) indicate that they are responsible for the induction of primary immunity. Two subclasses of DC have been recently identified in spleen that differ in their phenotype and in certain regulatory features. In vitro, both subsets have the capacity to activate naive T cells, although CD8a+ DC have been shown to induce T cell apoptosis and to stimulate lower levels of cytokines compared with CD8alpha- DC. The objective of this study was to analyze the function of these distinct DC types in vivo. Our results show that both subsets, pulsed extracorporeally with antigen and injected in the footpads of syngeneic mice, sensitize an antigen-specific T cell primary response. However, CD8alpha+ cells trigger the development of Thl-type cells, whereas CD8alpha- DC induce a Th2-type response. These observations suggest that the Th1/Th2 balance in vivo is regulated by the antigen-presenting-cells of the primary immune responses.
Asunto(s)
Antígenos CD8/inmunología , Células Dendríticas/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Diferenciación Celular , Humanos , Inmunofenotipificación , Interleucina-12/inmunología , Ligandos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células TH1/citología , Células TH1/inmunología , Células Th2/citología , Células Th2/inmunologíaRESUMEN
Injection of a staphylococcal superantigen (SAg) such as staphylococcal enterotoxin B (SEB) in adult mice results in cytokine production and cell proliferation which can lead to septic shock. The aim of the present work was to identify the cytokines and co-stimulatory molecules regulating the in vivo systemic release of IFN-gamma, a cytokine known to play an important role in the pathophysiology associated with bacterial infections. We demonstrate in this study that (i) in contrast to lipopolysaccharide (LPS), SEB administration induces high levels of the p70, bioactive form, of IL-12; (ii) IL-12 production in response to SEB requires both CD40-dependent signals and IFN-gamma secretion; (iii) the early systemic release of IFN-gamma (3 h post-treatment) in response to SEB is IL-12 independent, while the sustained, late response (6-9 h post-treatment) requires endogenous IL-12 production; (iv) IL-12 produced during the primary SEB response (day 0) is responsible for priming cells in vivo to high IFN-gamma production upon secondary challenge (day 2); (v) the priming effect of IL-12 is TCR unrelated, as SEB-primed animals secrete high levels of IFN-gamma in response to both staphylococcal enterotoxin A and LPS administered 48 h later. The ability of bacterial SAg to induce septic shock and to modulate the immune response to unrelated antigens may therefore be related to their unique capacity to induce systemic IL-12 production in vivo. These observations also help to explain why SEB-primed animals, known to express an anergic phenotype 48 h post-treatment (as judged by defective IL-2 production and proliferation), nevertheless display an increased capacity to secrete the inflammatory cytokine IFN-gamma.
Asunto(s)
Enterotoxinas/farmacología , Interferón gamma/metabolismo , Interleucina-12/fisiología , Superantígenos/farmacología , Animales , Dimerización , Regulación hacia Abajo , Enterotoxinas/administración & dosificación , Ensayo de Inmunoadsorción Enzimática , Femenino , Interleucina-12/farmacología , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Organismos Libres de Patógenos Específicos , Superantígenos/administración & dosificaciónRESUMEN
Cells of the dendritic family display some unique properties that confer to them the capacity to sensitize naive T cells in vitro and in vivo. In the mouse, two subclasses of dendritic cells (DCs) have been described that differ by their CD8alpha expression and their localization in lymphoid organs. The physiologic function of both cell populations remains obscure. Studies conducted in vitro have suggested that CD8alpha+ DCs could play a role in the regulation of immune responses, whereas conventional CD8alpha- DCs would be more stimulatory. We report here that both subclasses of DCs efficiently prime antigen-specific T cells in vivo, and direct the development of distinct T helper (Th) populations. Antigen-pulsed CD8alpha+ and CD8alpha- DCs are separated after overnight culture in recombinant granulocyte/macrophage colony-stimulating factor and injected into the footpads of syngeneic mice. Administration of CD8alpha- DCs induces a Th2-type response, whereas injection of CD8alpha+ DCs leads to Th1 differentiation. We further show that interleukin 12 plays a critical role in Th1 development by CD8alpha+ DCs. These findings suggest that the nature of the DC that presents the antigen to naive T cells may dictate the class selection of the adaptative immune response.